antioxidant, cytotoxic and dna protective properties of

AJP, Vol. 7, No. 2, Mar-Apr 2017 157

Original Research Article

Antioxidant, cytotoxic and DNA protective properties of Achillea

eriophora DC. and Achillea biebersteinii Afan. extracts: A comparative

study

Maryam Varasteh-kojourian1, Parvaneh Abrishamchi

1*, Maryam M. Matin

1,2, Javad Asili

3,

Hamid Ejtehadi1, Fatemeh Khosravitabar

1

1Department of Biology, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran

2Cell and Molecular Biotechnology Research Group, Institute of Biotechnology, Ferdowsi University of

Mashhad, Mashhad, Iran 3Department of Pharmacognosy, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

Article history: Received: Sep 24, 2015 Received in revised form: Feb 15, 2016 Accepted: May 08, 2016 Vol. 7, No. 2, Mar-Apr 2017, 157-168.

* Corresponding Author: Tel: +989151246726 Fax: +985138796416 [email protected]

Keywords: Phenol Flavonoids DPPH MTT assay Comet HFF3 cells

Abstract Objective: Achillea is a traditional medicinal herb which contains different phenol and flavonoid compounds that are responsible for Achillea pharmacological effects. We aimed to determine phenol and flavonoid contents, besides antioxidant activities of different extracts from Achillea eriophoraa (A. eriophora) DC. and Achillea biebersteinii (A. biebersteinii) Afan. (endemic species in Iran) and to investigate their effects on human cells. Materials and Methods: Achillea extracts, were prepared by maceration and shaking methods, from different parts (aerial parts, stem, leaves and inflorescence) of two species using methanol and ethanol as solvents. Total phenol and flavonoid contents were measured by spectrophotometry, and antioxidant activities of the extracts were determined by DPPH radical scavenging, BCB and TBARS assays. Cytotoxicity and antioxidant activities of the extracts were investigated in Human Foreskin Fibroblast (HFF3) cells using MTT, comet and H2O2 assays. Results: Methanol extracts of A. biebersteinii prepared from leaves and inflorescence by maceration method exhibited maximum phenol (1657.58 ± 36.45 mg GAE/100 g DW) and flavonoid (264.00 ± 62.16 mg QUE/100 g DW) contents. Leaf methanol extract showed significantly higher antioxidant activity (0.0276 ± 0.003, 0.16 ± 0.016 and 13.96 ± 0.26 mg/ml for DPPH, BCB and TBARS IC50s, respectively) than those of the other extracts. Leaf extract of A. biebersteinii was not cytotoxic even at the highest examined dose (512 µg/ml) and inhibited cell toxicity induced by H2O2 (98% viability for the cells pretreated with plant extract in the presence of H2O2). Comet assay also confirmed high DNA protective activity of leaf extracts. Conclusion: Achillea extracts possess remarkable antioxidant activity, and could be good natural alternatives to synthetic antioxidants in pharmaceutical and food industries.

Please cite this paper as:

Varasteh-kojourian M, Abrishamchi P, Matin MM, Asili J, Ejtehadi H, Khosravitabar F. Antioxidant,

cytotoxic and DNA protective properties of Achillea eriophora DC. and Achillea biebersteinii Afan. extracts: A

comparative study. Avicenna J Phytomed, 2016. Epub ahead of print.

Varasteh-kojourian et al.

AJP, Vol. 7, No. 2, Mar-Apr 2017 158

Introduction Achillea, which belongs to the family

Compositae (Asteraceae), is a genus with

over 100 species all around the world.

Although these medicinal perennial

rhizomatous plants are native to Europe

and Western Asia, they are also found in

Australia, New Zealand and North

America (Chevallier, 2000). The two

studied species Achillea eriophora (A.

eriophora) DC. And Achillea biebersteinii

(A. biebersteinii) Afan. are endemic plants

in Iran.

Achillea, known as “Bumadaran” in

Persian, is one of the most widely used

medicinal plants in Iran. It is used as

hypoglycemic, nerve tonic, anti-

hemorrhoid, anti-diarrhea, antacid,

carminative, appetizer, anthelmintic and

anti-bacterial remedies (Amiri and

Joharchi, 2013; Ghorbani, 2005; Pirbalouti

and Golparvar, 2007; Zargari, 1993).

These pharmacological properties have

been mainly attributed to the phenolic and

polyphenolic compounds, which are well

known as antioxidant agents (Evans, 2009;

Harborne and Williams, 2000; Weber et

al., 2006). Clinical evidences have

revealed that antioxidants are effective in

the treatment of various diseases, including

atherosclerosis, arthritis, ischemia and

reperfusion injury of many tissues, central

nervous system injury, gastritis, and

cancer, and are beneficial to the wound

healing process (Cook and Samman, 1996;

Kumpulainen and Salonen, 1999). These

activities are in accordance with reported

pharmaceutical properties for Achillea.

Antioxidant activity of different species

of Achillea including Achillea millefolium

L. (Candan et al., 2003), Achillea ligustica

All. (Conforti et al., 2005), Achillea

wilhelmsii (Ozgen et al. 2004) and A.

biebersteinii (Tawaha et al., 2007) have

been previously investigated. However, the

protective effects of A. eriophorea and A.

biebersteinii against oxidative stress in

HFF3 cells have not yet been reported. In

this study, two extraction methods and

solvents were used to obtain phenol and

flavonoid-enriched extracts from different

parts of A. eriophorea and A. biebersteinii.

The antioxidant activity of the methanol

extracts prepared by maceration from

different parts of the plants, was evaluated

using different assays. To the extent of our

knowledge, this is the first report about the

antioxidant activity of leaf methanol

extracts of A. eriophorea and A.

biebersteinii in human fibroblast cells.

Materials and Methods Chemicals

Folin-Ciocaltue, sodium carbonate,

methanol, ethanol, gallic acid, aluminum

chloride, 1,1-diphenyl-2-picrylhydrazyl

(DPPH), beta-carotene, linoleic acid,

tween40, chloroform, butylated hydroxyl

toluene (BHT), 2,2’-azobis-(2-

amidinopropane) dihydrochloride (ABAP),

acetic acid, tiubarbituric acid, sodium

dodecyl sulfate (SDS), butanol, 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyl-

tetrazolium bromide (MTT), H2O2,

ethylenediaminetetraacetic acid (EDTA),

dimethyl sulfoxide (DMSO) and all

solvents were purchased from Merck

(Germany). Ascorbic acid and potassium

acetate were purchased from Sigma–

Aldrich (St. Louis, MO, USA). Dulbecco's

Modified Eagle's Medium (DMEM) and

fetal bovine serum (FBS) were obtained

from Gibco Life Technologies (Grand

Island, NY, USA).

Sample collection and extract

preparation

Plants were collected at the flowering

stage in May 2011 from pastures of

Khorassan Razavi (N 36.291886, E

58.583121; 1618 m above sea level) and

South Khorasan provinces (N 36.34020, E

40.710158; 1591 m above sea level), Iran.

The voucher specimens (No. 30348 and

22004 for A. eriophora and A.

biebersteinii, respectively) were deposited

in Herbarium of Ferdowsi University,

Mashhad, Iran.

Antioxidant and cytotoxic properties of Achillea

AJP, Vol. 7, No. 2, Mar-Apr 2017 159

Grinded dry materials from different

parts of plants (stem, leaves, aerial parts

and inflorescence), were extracted with

methanol and ethanol (1:20 w/v) using

maceration and shaking. Extracts were

filtered through the regular filter paper and

evaporated under vacuum.

Total phenol and flavonoid assay

The total phenolic content of all extracts

was determined spectrophotometrically

according to Folin-Ciocalteu method

(Pattanayak et al., 2012), and total

flavonoid content was determined using

aluminum chloride colorimetric method

(Chang et al., 2002). The results were

expressed in terms of gallic acid equivalent

(GAE mg/g of dry weight) for phenolic

content and quercetin equivalent (QE mg/g

of dry weight) for flavonoid content,

which are the two common reference

compounds.

Antioxidant assays

DPPH radical scavenging microplate

assay

The antioxidant properties of methanol

extracts (0.5-7 mg/ml) or gallic acid as a

standard, were investigated by reducing

the stable DPPH radical (Yang et al.,

2011). The absorbance was then measured

at 492 nm using an ELISA reader

(Awareness Technology, USA). The

antioxidant index (AI %) was calculated as

[(1 – A1 – A2/ A0) × 100]. Where A0 is the

absorbance of the control reaction (without

sample), A1 is the absorbance of

sample/gallic acid, and A2 is the

absorbance of sample without DPPH.

Analyses were run in triplicate, and IC50

values (presenting the concentration with

50% antioxidant index) were calculated.

Beta-carotene bleaching microplate

assay (BCB)

A modified method of Dapkevicius et

al. (1998) was used to test bleaching

ability of the extracts against beta-carotene

(Dapkevicius et al., 1998). Briefly, 1 mg of

beta-carotene was dissolved in chloroform

(5 ml) and then, linoleic acid (25 µl) and

Tween 40 (200 mg) were added to 1 ml of

this mixture. After removing chloroform

using a rotary evaporator at 40ºC, the

remaining where solved in oxygenated

distilled water (50 ml) and vigorously

shacked. An aliquot of 250 µl of the

above-prepared beta carotene–linoleic acid

emulsion was applied to each well of a 96-

well plate. Next, 30 µl of different

concentrations of the extracts (0.5-7

mg/ml) or BHT as a standard (1-100

µg/ml) were added to each well in

triplicate. An equal amount of the extracts

or BHT was used as blank. The

microplates were incubated at 55ºC and

their optical densities were determined at

492 nm using an ELISA reader. Reading

the absorbance of all samples was carried

out at the start (t=0) and after 105 min of

incubation. The antioxidant activity

coefficient (AAC) was estimated

according to the following formula:

ACC = [(AT105 - AB105) / (AB0–AB105)]

Where AT105 and AB105 are absorbance

of sample and blank after 105 min,

respectively. AB0 is the absorbance of

blank at t=0.

TBARS assay

A modified TBARS assay (Bazzaz et

al., 2011), using egg yolk homogenates as

lipid rich media, was applied to measure

the antioxidant capacity of the extracts.

Briefly, 500 µl of yolk homogenate (10%

w/v in distilled water) and 100 µl of

sample solution (5-50 mg/ml

concentrations of the extracts or standard),

were added to a test tube and made up to 1

ml with distilled water, followed by

addition of 50 µl of ABAP aqueous

solution (0.07 M; for induction of lipid

peroxidation), 1.5 ml of 20% acetic acid

(pH 3.5) and 1.5 ml of 0.8% (w/v)

thiobarbituric acid in 1.1% (w/v) SDS. The

mixture was vortexed, and heated at 95ºC

for 60 min. After cooling, 5 ml butan-1-ol

was added and extensively vortexed and

centrifuged at 2500 g for 10 min. The

absorbance of the upper organic layer was


AJP, Vol. 7, No. 2, Mar-Apr 2017 160

measured at 532 nm using a

spectrophotometer. Butanol and BHT were

used as the blank and positive control,

respectively. All testes were carried out in

triplicate. Values were expressed as

antioxidant index (AI%) according to the

following formula:

AI% = (1 – t / c) × 100

Where t and c are the absorbance of the

test sample and the fully oxidized control,

respectively.

Cell culture

Cytotoxicity assay

HFF3 (Human Foreskin Fibroblast)

cells (a generous gift from Royan Institute,

Tehran, Iran) were seeded at 8000

cells/well in 96-well plates in DMEM

supplemented with 10% FBS. After 24 h

of incubation in a humidified 5% CO2/air

environment at 37ºC, when cells became

70-80% confluent, they were treated with

different concentrations of the extracts (1-

512 µg/ml) diluted in DMEM containing

1% FBS. Following 24, 48 and 72 h of

incubation with the extracts, culture media

were aspirated and replaced with DMEM

containing 1% FBS and 20 µl of MTT

solution (0.5 mg/ml). After 4 h of

incubation, media were removed and

purple colored crystals were dissolved in

DMSO. Absorbance of each well was

measured at 450 nm using an ELISA

reader.

Antioxidant activity of plant extracts on

HFF3 cells

The hydrogen peroxide (H2O2) assay

was modified to assess the protective

effects of the extracts in HFF3 cells

against the oxidative damage induced by

H2O2 (Kumar and Gupta, 2011). Fibroblast

cells were seeded in 96-well plates at 8000

cells/well in DMEM containing 10% FBS,

and grown at 37ºC to near confluence.

Cells were serum-deprived and pretreated

with leaf extracts 1 µg/ml for 2 h.

Pretreated cells were then exposed to

different concentrations of H2O2 (10, 100,

250, 500 and 1000 mM), for 24 h. The

degree of protection of fibroblast cells by

extracts against H2O2 damage was then

quantified by MTT assay.

Comet assay

Single cell gel electrophoresis was

performed (Rassouli et al., 2011) on HFF3

cells treated with H2O2 and leaf extracts.

HFF3 cells were grown in DMEM

containing 10% FBS in 6-well plates, for

24 h. The attached cells were pretreated for

2 h with 5 ml DMEM (1% FBS)

containing leaf extracts 1 µg/ml of both

species. After this pretreatment, H2O2 (500

mM) was added and cells were incubated

for 24 h. Cells were then washed with 5 ml

cold PBS, and detached by trypsin/EDTA

solution for further analysis. Each data

point represents the average DNA in tail of

at least 150 measurements (comets).

Comets were analyzed with Tri Teck

Comet Score version 1.5.

Statistical analysis

Phenol and flavonoid contents were

analyzed using univariate ANOVA, and

Duncan as multiple range mean comparing

test. One-way ANOVA was used to

analyze the IC50 values of antioxidant

testes, means of H2O2 and comet assay

results. Also, Duncan was performed as

post-hoc analysis. Results were expressed

as mean ± S.D. All analyses were

performed using STATISTICA (Statsoft,

2011) software.

Results Total phenol and flavonoid contents

Total phenol and flavonoids content in

the Achillea species ranged from 149 to

1657 mg gallic acid equivalent (GAE) /100

g dry weight, and 59-264 mg quercetin

equivalent (QUE) /100 g dry weight,

respectively (Table 1). The highest total

phenol content was shown by

inflorescence extract followed by leaf

extract of A. biebersteinii. Also, the

highest total flavonoid content was

recorded for leaves extract of both species.


AJP, Vol. 7, No. 2, Mar-Apr 2017 161

Methanol extracts prepared by maceration

method, possessed higher phenol and

flavonoid content as compared to those

prepared with ethanol and shaking

methods.

Table 1. Effect of extraction methods (maceration and shaking) and solvent type on total phenol and total

flavonoid content in different parts of Achilea eriophora and Achilea biebersteinii from Iran.

In each row, presented data belong to maceration and shaking methods, respectively. *, $ p<0.05. Data are

means ± SD of five replicates.

Evaluation of antioxidant activities by

different methods

Since among all the extracts, leaf and

inflorescence methanol extract showed the

highest phenol and flavonoid contents,

maceration extraction and methanol

solvents were selected for evaluation of

antioxidant activities. The antioxidant

capacity of Achillea extracts, as presented

in Table 2, were determined by the

following three methods: DPPH, BCB and

TBARS assays. According the three

different assays, leaf extract of A.

biebersteinii, showed the highest

antioxidant activity in three selected

assays. IC50 values for leaf extract of A.

biebersteinii were 0.27, 0.16 and 13.96

mg/ml, in DPPH, BCB and TBARS tests,

respectively. Inflorescence extract of A.

eriophora showed the lowest DPPH

radical scavenging activity. Also, at

concentrations below 100 mg/ml of

inflorescence extracts in both species,

TBARS AI did not reach 50%.

Effects of Achillea leaf extracts on

viability of HFF3 cells

Antioxidant capacity and cytotoxic

effect of methanol extracts of Achillea

leaves were examined in vitro. Viability of

HFF3 cells treated with different

concentrations of A. eriophora extracts,

Plant organ Solvent Total phenol

mg GAE/100 g dry plant tissue

Total flavonoid

mg QUE/100 g dry plant tissue

Stem (A. eriophora)

methanol 316.79 ± 43.32 92.13±4.02

278.02 ± 110.33 88.12±8.48

ethanol 229.93 ± 9.02 70.04±2.75

225.37 ± 37.59 59.25±4.30

Leaves

methanol 1050.82 ± 184.20 244.06±1.27

890.17 ± 75.27 232.08±8.23

ethanol 622.76 ± 138.90

740.92 ± 84.48

226.42±1.71

196.58±6.71

Aerial parts

methanol 667.74 ± 73.30 216.45±2.15

581.09 ± 98.45 201.69±44.36

ethanol 327.15 ± 46.80 93.81±2.89

365.29 ± 142.42 81.83±12.99

Inflorescence

methanol 812.23 ± 64.39 216.56±1.67

807.46 ± 105.01 190.13±16.35

ethanol 570.72 ± 51.53 156.97±4.88

566.16 ± 55.75 133.89±10.76

Stem (A.biebersteinii)

methanol 293.36 ± 77.24 77.79±4.40

284.45 ± 51.04 74.00±4.09

ethanol 149.91 ± 16.39 74.19±7.17

164.84 ± 12.84 71.01±1.54

Leaves

methanol 1168.98 ± 146.10 249.99±12.01$

1156.34 ± 76.92 264.00±62.16$

ethanol 694.48 ± 56.81 236.36±3.25

719.15 ± 26.30 207.82±5.08

Aerial parts

methanol 848.30 ± 121.90 203.86±6.63

821.14 ± 51.98 210.21±21.79

ethanol 454.02 ± 57.88 214.91±4.00

389.13 ± 81.48 188.15±1.93

Inflorescence

methanol 1657.58 ± 36.45* 226.09±4.83

1441.79 ± 215.27 197.49±28.53

ethanol 1347.47 ± 61.37 184.99±8.13

1228.68 ± 50.85 169.25±6.73


AJP, Vol. 7, No. 2, Mar-Apr 2017 162

reduced in a dose-dependent manner, but

the highest examined dose (512 µg/ml) of

A. biebersteinii extract showed no toxic

effects. IC50 values obtained for A.

eriophora extract were 120, 85 and 55

mg/ml after 24, 48 and 72 h treatments,

respectively (Figures 1 and 2).

Table 2. Antioxidant activities of the Achillea extracts

Plant Species Plant organ IC50 (mg/ml)

DPPH BCB TBARS

Achillea

eriophora

Leaves 0.703±0.023 b 1.46±0.03 a 23.83±0.5 a

Inflorescence 0.91±0.001 a 1.78±0.441 a > 100

Achillea

biebersteini

Leaves 0.276±0.003 d 0.16±0.016 b 13.96±0.26 b

Inflorescence 0.33±0.006 c 1.63±0.176 a > 100

Ascorbate 0.016±0.0003 e - -

BHT - 0.00015±0.00002 b 4.93±0.74 c

Data are mean ± SD of three replicates. a-e, means in each column following different letters are significantly

different (p<0.05) as determined by Duncan’s multiple rang test.

Figure 1. The cytotoxicity of the methanol extract of the leaves of Achilea eriophora on the proliferation of

human foreskin fibroblast cells (HFF3). The cells were treated with various concentrations (1-512 µg/ml) of the

extract in a culture medium for 24, 48 and 72 h. Cells viability was determined and compared with the control

(untreated cells) by the MTT assay. Each value represents the mean ± SD (n=3). *p<0.05 Starred values, are

significantly different from other means.

Figure 2. The cytotoxicity of the methanol extract of the leaves of Achilea biebersteinii on the proliferation of

human foreskin fibroblast cells (HFF3). The cells were treated with various concentrations (1-512 µg/ml) of the

extract in a culture medium for 24, 48 and 72 h. Cells viability was determined and compared with the control

(untreated cells) by the MTT assay. Each value represents the mean ± SD (n=3). *p<0.05 Starred values, are

significantly different from other means.


AJP, Vol. 7, No. 2, Mar-Apr 2017 163

Antioxidant activity of leaf extracts on

HFF3 cells

Achillea leaf extracts were selected due

to their high phenol content and

antioxidant activity, and their possible

protective effects against H2O2-induced

damages were investigated in vitro. The

protective effects of the extracts on HFF3

cells were first confirmed by MTT assay.

Reduced viability was recorded at different

concentrations of H2O2 (54.73% at 250

mM H2O2, Figure 3), but pre-treatment

with 1 µg/ml of the Achillea leaf extracts,

could significantly (p<0.05) inhibit

oxidative damage (Figure 3). Also results

showed that pre-treatment with A.

biebersteinii leaves extract were more

effective than pre-treatment with A.

eriophorea to inhibit cell injuries caused

by H2O2 treatment (Figure 4). The

oxidative damage to the DNA of HFF3

cells was examined using comet assay in

H2O2-treated cells.

Figure 3. The effect of methanol extract on H2O2

cytotoxicity in human foreskin fibroblast cells

(HFF3). Cytotoxic effect of H2O2 at 0-1000 mM

concentrations (A) were compared to cells pre-

treated with 1 µg/ml A. biebersteinii leaves extract

(B). Cells viability (%) was determined and

compared with the control (untreated cells) using

MTT assay. Each value represents the mean ± SD

of three replications. *p<0.05 compared with the

corresponding concentration of H2O2 without plant

extract pre-treatment.

Figure 4. Achillea extracts inhibited HFF3 cells

oxidative injury. HFF3 cells injury induced by

H2O2 were inhibited by pretreatment with Achillea

extracts (1 µg/ml). The percentage of cell injury

inhibition was calculated on the basis of viability

using MTT assay. Data are mean ± SD of three

replicates. *p<0.05 compared with corresponding

concentration of H2O2 with different plant extract

pre-treatment.

Comet results also confirmed protective

effects of leaf extracts (1 µg/ml) against

H2O2-induced oxidative damage. As

shown in Figure 5 and 6, DNA in tail was

reduced in cells pretreated with Achillea

extracts after H2O2 treatment (8.9% and

7.6% for A. biebersteinii and A. eriophora,

respectively), as compared to H2O2-treated

cells in which the DNA in tail was about

66%.

Figure 5. Effect of methanol extracts from the

leaves of two Achillea species (1 µg/ml) on DNA

damage induced by H2O2 (500 mM) in human

foreskin fibroblast cells (HFF3) assessed by Comet

assay. A. eriophora and A. biebersteinii groups

were pre-treated with different extracts (1 µg/ml),

DMSO group was pretreated with DMSO and

untreated group was the null control without any

pretreatment. DNA in tail in each cell was

calculated by comet score. The data represent

mean±SD of 150 cells. *p<0.05 Starred values

compared with means in untreated and DMSO

groups which are treated with H2O2.


AJP, Vol. 7, No. 2, Mar-Apr 2017 164

Figure 6. Comet tails of human foreskin fibroblast

cells (HFF3) treated with H2O2 and Achillea

extracts. Pre-treatment with the methanol extract of

Achillea leaves (1 µg/ml) significantly (p<0.05)

reduced comet tail of HFF3 cells induced by H2O2

(500 mM) treatment. Untreated cells (a), H2O2

treated cells (b), H2O2-incubated cells pretreated

with Achillea eriophora (c), and A. biebersteinii

extracts (d). Arrows indicate comet tails which

contain damaged DNA.

Discussion There are different reports on the total

phenolic contents of the leaf extract of A.

biebersteinii. For example, Tawaha et al.

reported that this value varied between

1600 and 2300 mg of GAE/100 g (Tawaha

et al., 2007), while in the study of Sokmen

and Özbek, it was calculated as 51 ± 0.6

mg/g (Sokmen and Özbek, 2006). These

differences may be mainly due to

diversities of the analytical methods,

extraction methods, variety, developmental

phase, and geographic origin of the plants.

Similar to our results (Table 1), there is a

study that reported the total phenol

contents of A. eriophora to be were higher

in leaves than inflorescence (Dokhani et

al., 2005).

As indicated in the results, leaf extracts

of A. biebersteinii with the highest

antioxidant activity (as measured by three

different methods), showed the highest

phenol and flavonoid contents as well. It

seems that higher antioxidant activity of A.

biebersteinii extracts, as compared to A.

eriophora extracts, could be attributed to

their higher phenolic and flavonoid

contents.

This study demonstrated that the

cytotoxicity induced via oxidative stress

(H2O2-induced stress) could be suppressed

by pre-treatment with Achillea leaf

extracts (1μg/ml). Protective effects of

plant extracts against oxidative cell

damage, have been previously reported by

various researchers (Gião et al., 2010;

Konyalioglu and Karamenderes, 2005;

Yoo et al., 2008). Adetutu et al. (2011) and

Annan and Houghton (2008) reported that

fibroblast cells were protected against

H2O2-induced oxidative damage using

Bridelia ferruginea Benth. and Gossypium

arboretum L., and Ficus asperifolia Miq.

Extracts (Adetutu et al., 2011; Annan and

Houghton, 2008). Adetutu et al. reached

82% protection, when the concentration of

plant extract was 250 µg/ml (in the

presence of 180 µM H2O2 as an oxidant),

and Annan and Houghton reported 58%

protection against oxidative damage at 50

µg/ml of F. asperifolia extract (in the

presence of 100 µM H2O2); However, we

observed 35.94% protection at a low

concentration (1 µg/ml) of A. biebersteinii

leaf extract (in the presence of 100-500

mM H2O2).

Anti-superoxide properties of flower

infusion of A. biebersteinii in erythrocytes

and leucocytes were reported. Cellular

damages induced by 10 mM H2O2, were

blocked after treatment with 500 µl of the

infusion. Phenol and flavonoid compounds

of infusion were responsible for the

positive effects on enzymatic antioxidant

system (Konyalioglu and Karamenderes,

2005). Behravan et al. (2011) reported a

significant inhibitory effect of aqueous

extract of Portulaca oleracea L. at 1 and

2.5 mg/ml on H2O2-induced DNA damage

in human lymphocyte (percentage tail

DNA 2.35% ± 0.16 and 1.29% ± 0.12,

respectively). Pretreatment with Mentha

arvensis L. (25 μg/ml), exhibited a

significant protective effect (12.43% of tail

DNA% compared to the control) in

lymphocytes (Lin et al., 2013). As Achillea


AJP, Vol. 7, No. 2, Mar-Apr 2017 165

species are effective antioxidants,

according to the result of antioxidant

assays (DPPH, BCB, TBARS), our results

suggest that it is likely that the inhibitory

effect of Achillea on H2O2-induced DNA

damage could be the result of interactions

of different antioxidant compounds in the

extracts, as reported for P. oleracea and M.

arvensis (Behravan et al., 2011; Lin et al.,

2013).

Although this study revealed

remarkable antioxidant properties of A.

biebersteinii and A. eriophora extracts,

further studies are required to determine

the chemical composition of the extracts

and antioxidant activities of phenolic acids

and flavonoids, as their main constituents.

Other obvious important topics of research

will be to determine the most effective

compounds and their mode of action in

cell-based assays.

A. eriophora and A. biebersteini, with

remarkable antioxidant activities,

especially on human fibroblast cell culture,

could be considered as good sources of

natural antioxidants and healthy

replacements for the corresponding

synthetic ones such as industrial food

preservatives. Their high phenol and

flavonoid contents might be responsible

for the traditional usage of these medicinal

plants; however, we recommend more

detailed studies to determine their exact

chemical composition and mechanism of

action.

Acknowledgments

The authors appreciate the financial

support provided by Ferdowsi University

of Mashhad [number 3/16744].

Conflict of interest The authors report no declarations of

interest.

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